A Frameshift Mutation in GON4L Is Associated with Proportionate Dwarfism in Fleckvieh Cattle
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bioRxiv preprint doi: https://doi.org/10.1101/036889; this version posted March 9, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. A frameshift mutation in GON4L is associated with proportionate dwarfism in Fleckvieh cattle Hermann Schwarzenbacher1, Christine Wurmser2, Krzysztof Flisikowski3, Lubica Misurova4, Simone Jung2,*, Martin C. Langenmayer5,#, Angelika Schnieke3, Gabriela Knubben-Schweizer4, Ruedi Fries2, Hubert Pausch2,$ 1 ZuchtData EDV-Dienstleistungen GmbH, 1200 Vienna, Austria 2 Chair of Animal Breeding, Technische Universitaet Muenchen, 85354 Freising, Germany 3 Chair of Animal Biotechnology, Technische Universitaet Muenchen, 85354 Freising, Germany 4 Clinic for Ruminants with Ambulatory and Herd Health Services at the Centre for Clinical Veterinary Medicine, Ludwigs-Maximilians-Universitaet Muenchen, 85764 Oberschleissheim, Germany 5 Institute of Veterinary Pathology at the Centre for Clinical Veterinary Medicine, Ludwigs-Maximilians-Universitaet Muenchen, 80539 Muenchen, Germany * present address: Bayern Genetik GmbH, 85586 Poing, Germany # present address: Institute for Infectious Diseases and Zoonoses, Ludwigs- Maximilians-Universitaet Muenchen, 80539 München $ corresponding author: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/036889; this version posted March 9, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Abstract Background Low birth weight and postnatal growth restriction are the most evident symptoms of dwarfism. Accompanying skeletal aberrations may compromise the general condition and locomotion of affected individuals. Several paternal half sibs with low birth weight and size were born in 2013 in the Fleckvieh cattle population. Results Affected calves were strikingly underweight at birth despite a normal gestation length and had craniofacial abnormalities such as elongated narrow heads and brachygnathia inferior. Despite a normal general condition, their growth remained restricted during rearing. We genotyped 27 affected and 10,454 unaffected animals at 44,672 SNPs and performed association testing followed by homozygosity mapping to map the growth failure to a 1.85 Mb segment on bovine chromosome 3. Analysis of whole-genome re- sequencing data from one affected and 289 unaffected animals revealed a 1bp deletion (g.15079217delC, rs723240647) in the coding region of the GON4L gene that segregated with the dwarfism-associated haplotype. We show that the deletion introduces intron retention and premature termination of translation, putatively resulting in a severely truncated protein that lacks domains that are likely essential to normal protein function. The widespread use of an unnoticed carrier bull for artificial insemination has resulted in a tenfold increase in the frequency of the deleterious allele in the female population. Conclusions A frameshift mutation in GON4L is associated with autosomal recessive proportionate dwarfism in Fleckvieh cattle. The mutation has segregated in the population for more than fifty years without being recognized as a genetic disorder. However, the widespread use of an unnoticed carrier bull for artificial insemination caused a sudden accumulation of homozygous calves with dwarfism. Our findings provide the basis for genome-based mating strategies to avoid the inadvertent mating of carrier animals and thereby prevent the birth of homozygous calves with impaired growth. 2 bioRxiv preprint doi: https://doi.org/10.1101/036889; this version posted March 9, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Background Bovine stature is a prototypical complex trait that is controlled by a few loci with large effects and numerous loci with small effects. Genome-wide association studies using dense molecular markers revealed several quantitative trait loci (QTL) for growth-related traits in cattle [1-3]. The identified QTL account for a reasonable fraction of the phenotypic variation of bovine height [2, 4]. Sequence variants associated with mature height may also affect the size and weight of newborn calves [2, 3, 5]. Birth size and weight varies between breeds, parities and the sex of the calf [6, 7]. The birth weight in Fleckvieh cattle typically ranges from 38 to 45 kg [8]. Calves with strikingly low birth weight and size despite normal gestation length are commonly referred to as “dwarfs”. Dwarfism (DW) has been observed in several cattle breeds including Fleckvieh [9- 11]. Low birth size and postnatal growth restriction are the most apparent characteristics of DW. Undersized animals may be normally proportionated and have an undisturbed general condition (i.e., proportional DW [12]). However, DW may also be accompanied by disproportionately shortened limbs and skeletal deformities (i.e., disproportional DW, chondrodysplasia [13]). Depending on the severity of the structural aberrations, disproportionate DW may be fatal [14, 15]. Both autosomal recessive and dominant inheritance has been reported for bovine DW (e.g., [12, 15]). Causative mutations for DW were identified in Angus (OMIA 001485-9913 [16]), Dexter (OMIA 001271-9913 [14]), Tyrolean Grey (OMIA 000187-9913 [13]), Holstein-Friesian (OMIA 001926-9913 [15]) and Japanese Brown cattle (OMIA 000187-9913 [17]). However, mutations causing DW have not yet been identified in Fleckvieh cattle. Here we present the phenotypic and genetic characterization of autosomal recessive DW in Fleckvieh cattle. The use of genome-wide association testing, autozygosity mapping and massive re-sequencing data enabled us to identify a frameshift mutation in GON4L that is likely causal for the growth failure. 3 bioRxiv preprint doi: https://doi.org/10.1101/036889; this version posted March 9, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Methods Animal ethics statement Two animals were hospitalized at the animal clinic of Ludwigs-Maximilians- Universität München. Another two animals were pathologically examined at the Institute for Veterinary Disease Control (IVDC) of Austrian Agency for Health and Food Safety. One hospitalized calf was euthanized because of recurrent tympania with no prospect of improvement and subsequently necropsied. Tissue samples were collected during necropsy. All affected animals result from inadvertent carrier matings that happened in Fleckvieh farms. No ethical approval was required for this study. Animals Twenty-seven paternal half sibs (16 male, 11 female) with strikingly low birth weight and postnatal growth restriction were inspected by breeding consultants at the age of three weeks to 18 months. Ear tissue samples were collected by breeding consultants and DNA was prepared following standard DNA extraction protocols. Genotyping, quality control and haplotype inference Twenty-seven affected animals were genotyped with the Illumina BovineSNP50 v2 BeadChip interrogating genotypes at 54,609 SNPs. The per-individual call-rate ranged from 98.96% to 99.60% with an average call-rate of 99.33%. In addition, genotypes of 10,454 unaffected Fleckvieh animals were available from genotyping with the Illumina BovineSNP50 v1 BeadChip and the Illumina BovineHD BeadChip [18, 19]. The genotype data of cases and controls were combined and SNPs that were present in both datasets were retained for further analyses. Following quality control (minor allele frequency above 0.5%, no deviation from the Hardy-Weinberg equilibrium (P>0.0001), per-SNP and per-individual call-rate higher than 95%), 10,481 animals (27 affected, 10,454 unaffected) and 44,672 SNPs remained for association testing. The Beagle software [20] was used to impute sporadically missing genotypes and to infer haplotypes. Haplotype-based association testing 4 bioRxiv preprint doi: https://doi.org/10.1101/036889; this version posted March 9, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. A sliding window consisting of 25 contiguous SNPs (corresponding to an average haplotype length of 1.42±0.43 Mb) was shifted along the genome in steps of 2 SNPs. Within each sliding window, all haplotypes with a frequency above 0.5% (N=787,232) were tested for association with the affection status using Fisher exact tests of allelic association. Haplotypes with a P value less than 6.35 x 10-8 (5 % Bonferroni-corrected significance threshold) were considered as significantly associated. Generation of sequence data Genomic DNA was prepared from a frozen semen sample of the supposed founder (DWhet) and from an ear tissue sample of one affected animal (DWhom) following standard DNA extraction protocols. Paired-end libraries were prepared using the paired-end TruSeq DNA sample preparation kit (Illumina)